Liver fibrosis is a dynamic wound-healing response characterized by the agglutination of the extracellular matrix (ECM). Si-Wu-Tang (SWT), a traditional Chinese medicine (TCM) formula, is known for treating gynecological diseases and liver fibrosis. Our previous studies demonstrated that long non-coding RNA H19 (H19) was markedly upregulated in fibrotic livers while its deficiency markedly reversed fibrogenesis. However, the mechanisms by which SWT influences H19 remain unclear. Thus, we established a bile duct ligation (BDL)-induced liver fibrosis model to evaluate the hepatoprotective effects of SWT on various cells in the liver. Our results showed that SWT markedly improved ECM deposition and bile duct reactions in the liver. Notably, SWT relieved liver fibrosis by regulating the transcription of genes involved in the cytoskeleton remodeling, primarily in hepatic stellate cells (HSCs), and influencing cytoskeleton-related angiogenesis and hepatocellular injury. This modulation collectively led to reduced ECM deposition. Through extensive bioinformatics analyses, we determined that H19 acted as a miRNA sponge and mainly inhibited miR-200, miR-211, and let7b, thereby regulating the above cellular regulatory pathways. Meanwhile, SWT reversed H19-related miRNAs and signaling pathways, diminishing ECM deposition and liver fibrosis. However, these protective effects of SWT were diminished with the overexpression of H19 in vivo. In conclusion, our study elucidates the underlying mechanisms of SWT from the perspective of H19-related signal networks and proposes a potential SWT-based therapeutic strategy for the treatment of liver fibrosis.
Humans ; RNA, Long Noncoding/genetics* ; Liver Cirrhosis/genetics* ; Liver/metabolism* ; Hepatic Stellate Cells/pathology* ; MicroRNAs/metabolism* ; Extracellular Matrix/metabolism* ; Drugs, Chinese Herbal

Objective: To explore the pathogenic mechanism of the miR-340/high mobility group box 1 (HMGB1) axis in the formation of liver fibrosis. Methods: A rat liver fibrosis model was established by injecting CCl(4) intraperitoneally. miRNAs targeting and validating HMGB1 were selected with gene microarrays after screening the differentially expressed miRNAs in rats with normal and hepatic fibrosis. The effect of miRNA expressional changes on HMGB1 levels was detected by qPCR. Dual luciferase gene reporter assays (LUC) was used to verify the targeting relationship between miR-340 and HMGB1. The proliferative activity of the hepatic stellate cell line HSC-T6 was detected by thiazolyl blue tetrazolium bromide (MTT) assay after co-transfection of miRNA mimics and HMGB1 overexpression vector, and the expression of extracellular matrix (ECM) proteins type I collagen and α-smooth muscle actin (SMA) was detected by western blot. Statistical analysis was performed by analysis of variance and the LSD-t test. Results: Hematoxylin-eosin and Masson staining results showed that the rat model of liver fibrosis was successfully established. Gene microarray analysis and bioinformatics prediction had detected eight miRNAs possibly targeting HMGB1, and animal model validation had detected miR-340. qPCR detection results showed that miR-340 had inhibited the expression of HMGB1, and a luciferase complementation assay suggested that miR-340 had targeted HMGB1. Functional experiments results showed that HMGB1 overexpression had enhanced cell proliferation activity and the expression of type I collagen and α-SMA, while miR-340 mimics had not only inhibited cell proliferation activity and the expression of HMGB1, type I collagen, and α-SMA, but also partially reversed the promoting effect of HMGB1 on cell proliferation and ECM synthesis. Conclusion: miR-340 targets HMGB1 to inhibit the proliferation and ECM deposition in hepatic stellate cells and plays a protective role during the process of liver fibrosis.
Animals ; Rats ; Cell Proliferation ; Collagen Type I/metabolism* ; Fibrosis ; Hepatic Stellate Cells ; HMGB1 Protein/genetics* ; Liver Cirrhosis/pathology* ; MicroRNAs/metabolism*

Fontan-associated liver disease (FALD) is one of the main complications after the Fontan procedure, manifesting mostly as liver fibrosis and even cirrhosis, with a high incidence rate and a lack of typical clinical symptoms that seriously affect patient prognosis. The specific cause is unknown, although it is considered to be associated with long-term elevated central venous pressure, impaired hepatic artery blood flow, and other relevant factors. The absence of association between laboratory tests, imaging data, and the severity of liver fibrosis makes clinical diagnosis and monitoring difficult. A liver biopsy is the gold standard for diagnosing and staging liver fibrosis. The most important risk factor for FALD is time following the Fontan procedure; therefore, it is recommended to do a liver biopsy 10 years after the Fontan procedure and to be cautious for the presence of hepatocellular carcinoma. Combined heart-liver transplantation is a recommended choice with favorable outcomes for patients with Fontan circulatory failure and severe hepatic fibrosis.
Humans ; Liver Diseases/pathology* ; Liver Cirrhosis/pathology* ; Liver/pathology* ; Carcinoma, Hepatocellular/pathology* ; Liver Transplantation/adverse effects* ; Fontan Procedure/adverse effects* ; Postoperative Complications/pathology* ; Liver Neoplasms/pathology*

An effective therapeutic regimen for hepatic fibrosis requires a deep understanding of the pathogenesis mechanism. Hepatic fibrosis is characterized by activated hepatic stellate cells (aHSCs) with an excessive production of extracellular matrix. Although promoted activation of HSCs by M2 macrophages has been demonstrated, the molecular mechanism involved remains ambiguous. Herein, we propose that the vitamin D receptor (VDR) involved in macrophage polarization may regulate the communication between macrophages and HSCs by changing the functions of exosomes. We confirm that activating the VDR can inhibit the effect of M2 macrophages on HSC activation. The exosomes derived from M2 macrophages can promote HSC activation, while stimulating VDR alters the protein profiles and reverses their roles in M2 macrophage exosomes. Smooth muscle cell-associated protein 5 (SMAP-5) was found to be the key effector protein in promoting HSC activation by regulating autophagy flux. Building on these results, we show that a combined treatment of a VDR agonist and a macrophage-targeted exosomal secretion inhibitor achieves an excellent anti-hepatic fibrosis effect. In this study, we aim to elucidate the association between VDR and macrophages in HSC activation. The results contribute to our understanding of the pathogenesis mechanism of hepatic fibrosis, and provide potential therapeutic targets for its treatment.
Humans ; Hepatic Stellate Cells/pathology* ; Receptors, Calcitriol ; Liver Cirrhosis/pathology* ; Macrophages/metabolism*

OBJECTIVE: Huangqi Decoction (HQD), a classical traditional Chinese medicine formula, has been used as a valid treatment for alleviating liver fibrosis; however, the underlying molecular mechanism is still unknown. Although our previous studies showed that microRNA-663a (miR-663a) suppresses the proliferation and activation of hepatic stellate cells (HSCs) and the transforming growth factor-β/small mothers against decapentaplegic (TGF-β/Smad) pathway, whether long noncoding RNAs (lncRNAs) are involved in HSC activation via the miR-663a/TGF-β/Smad signaling pathway has not yet reported. The present study aimed to investigate the roles of lncRNA lnc-C18orf26-1 in the activation of HSCs and the mechanism by which HQD inhibits hepatic fibrosis. METHODS: The expression levels of lnc-C18orf26-1, miR-663a and related genes were measured by quantitative reverse transcription-polymerase chain reaction. HSCs were transfected with the miR-663a mimic or inhibitor and lnc-C18orf26-1 small interfering RNAs. The water-soluble tetrazolium salt-1 assay was used to assess the proliferation rate of HSCs. Changes in lncRNA expression were evaluated in miR-663a-overexpressing HSCs by using microarray to identify miR-663a-regulated lncRNAs. RNA hybrid was used to predict the potential miR-663a binding sites on lncRNAs. Luciferase reporter assays further confirmed the interaction between miR-663a and the lncRNA. The expression levels of collagen α-2(I) chain (COL1A2), α-smooth muscle actin (α-SMA) and TGF-β/Smad signaling pathway-related proteins were determined using Western blotting. RESULTS: Lnc-C18orf26-1 was upregulated in TGF-β1-activated HSCs and competitively bound to miR-663a. Knockdown of lnc-C18orf26-1 inhibited HSC proliferation and activation, downregulated TGF-β1-stimulated α-SMA and COL1A2 expression, and inhibited the TGF-β1/Smad signaling pathway. HQD suppressed the proliferation and activation of HSCs. HQD increased miR-663a expression and decreased lnc-C18orf26-1 expression in HSCs. Further studies showed that HQD inhibited the expression of COL1A2, α-SMA, TGF-β1, TGF-β type I receptor (TGF-βRI) and phosphorylated Smad2 (p-Smad2) in HSCs, and these effects were reversed by miR-663a inhibitor treatment. CONCLUSION Our study identified lnc-C18orf26-1 and miR-663a as promising therapeutic targets for hepatic fibrosis. HQD inhibits HSC proliferation and activation at least partially by regulating the lnc-C18orf26-1/miR-663a/TGF-β1/TGF-βRI/p-Smad2 axis.
Humans ; Transforming Growth Factor beta/pharmacology* ; Transforming Growth Factor beta1/metabolism* ; RNA, Long Noncoding/pharmacology* ; Drugs, Chinese Herbal/pharmacology* ; MicroRNAs/genetics* ; Hepatic Stellate Cells/pathology* ; Liver Cirrhosis/metabolism* ; Cell Proliferation ; Transforming Growth Factors/pharmacology*

OBJECTIVE: To establish a visual reporting system for evaluating the activity of collagen Ⅰ α 1 chain (COL1A1) gene promoter in immortalized human hepatic stellate cells, so as to estimate the activation status of the cells and provide a new cell model for the screening and study of anti-hepatic fibrosis drugs. METHODS: The promoter sequence of human COL1A1 was amplified from the genomic DNA of human hepatocarcinoma cell line HepG2. Based on the pLVX-AcGFP1-N1 plasmid, the recombinant plasmid pLVX-COL1A1-enhanced green fluorescent protein (EGFP) was constructed, in which the enhanced green fluorescent protein gene expression was regulated by the COL1A1 promoter. The monoclonal cell line was acquired by stably transfecting pLVX-COL1A1-EGFP into the immortalized human hepatic stellate cell line LX-2 by the lentivirus packaging system and screening. The cell line was treated with transforming growth factor-β1 (TGF-β1) or co-treated with TGF-β1 and drugs with potential anti-hepatic fibrosis effects. The EGFP fluorescence intensity in cells was analyzed by the fluorescence microscope and ImageJ 1.49 software using a semi-quantitative method. The COL1A1 and EGFP mRNA were detected by reverse transcription real-time quantitative PCR (RT-qPCR), and corresponding proteins were detected by Western blot. RESULTS: The recombinant plasmid pLVX-COL1A1-EGFP with the expression of EGFP regulated by COL1A1 promoter was successfully constructed. Kozak sequence was added to enhance the expression of EGFP, which was identified by double digestion and sequencing. The LX-2 monoclonal cell line LX-2-CE stably transfected with pLVX-COL1A1-EGFP was obtained. After co-treatment with TGF-β1 and 5 μmol/L dihydrotanshinone Ⅰ with potential anti-hepatic fibrosis effect for 24 h, the total fluorescence intensity and the average fluorescence intensity of LX-2-CE were lower than those in TGF-β1 single treatment group (P < 0.05), the intracellular mRNA and protein levels of COL1A1 and EGFP were also lower than those in the TGF-β1 single treatment group (P < 0.05). CONCLUSION A reporter system for estimating activation of hepatic stellate cells based on COL1A1 promoter regulated EGFP expression is successfully constructed, which could visually report the changes in COL1A1 expression, one of the activation-related markers of hepatic stellate cells, in vitro. It provides a new cell model for the screening and study of anti-hepatic fibrosis drugs.
Humans ; Transforming Growth Factor beta1/pharmacology* ; Hepatic Stellate Cells/pathology* ; Liver Cirrhosis/genetics* ; Collagen Type I/pharmacology* ; RNA, Messenger/metabolism*

Primary myelofibrosis (PMF) is easily confused with cirrhosis, due to its main clinical manifestations of splenomegaly and the blood cytopenia. This review focuses on clinical studies to identify primary myelofibrosis and cirrhosis related portal hypertension, to analyze the differences between the two diseases, in order to distinguish PMF and cirrhosis from the pathogenesis, clinical manifestations, laboratory examinations and treatment principles, and simultaneously improve clinicians' understanding of PMF, which is a reference for exploring the early screening or diagnostic indicators of PMF, also provides a clinical basis for the application of new targeted drugs such as ruxolitinib.
Humans ; Primary Myelofibrosis/drug therapy* ; Hypertension, Portal/complications* ; Liver Cirrhosis/pathology* ; Splenomegaly/pathology* ; Anemia

OBJECTIVE: To explore the clinical effect of Li-Dan-He-Ji in the treatment of infantile cholestatic hepatic fibrosis. METHODS: Patients who met the diagnostic criteria of infantile cholestatic hepatic fibrosis in the department of integrated traditional Chinese and Western medicine and the department of gastroenterology of Wuhan Children's Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology from January to December 2021 were included in the study by prospective randomized controlled trial. They were divided into the conventional treatment group and Li-Dan-He-Ji group according to the random number table. The patients in the conventional treatment group were given conventional treatment according to the guidelines. In the Li-Dan-He-Ji group, the self-made Chinese medicinal compound Li-Dan-He-Ji (prescription: Herba Artemisiae Scopariae, Fructus Forsythiae, Radix et Rhizoma Rhei preparata, Radix Polygoni Multiflori Preparata, Radix Paeoniae Rubra, Ramulus Cinnamomi, Fructus Aurantii, Rhizoma Atractylodis Macrocephalae, Fructus Schisandrae Chinensis, Carapax Trionycis, and Radix Glycyrrhizae) was given on the basis of the routine treatment, by oral, enema or nasal feeding, 60 mL each day, divided into 2 or 3 times, for 28 days. Outpatient follow-up was maintained for 4 weeks. Before and after treatment, serum liver fibrosis 4 items [type IV collagen (IV-C), hyaluronidase (HA), type III procollagen (PC III), laminin (LN)], liver function and cholestasis-related markers [total bilirubin (TBil), direct bilirubin (DBil), total bile acid (TBA), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ-GGT), alanine aminotransferase (ALT), aspartate aminotransferase (AST)], oxidative stress markers [superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH)], liver stiffness measurement (LSM) detected by transient elastography (TE), aspartate aminotransferase-to-platelet ratio index (APRI), and liver and spleen retraction time were recorded in the two groups. RESULTS: During the observation period, a total of 40 cases of cholestatic hepatic fibrosis were treated, including 21 cases in the conventional treatment group and 19 cases in the Li-Dan-He-Ji group. Before treatment, the differences in serum liver fibrosis 4 items, serum liver function and cholestasis-related markers, oxidative stress indexes, LSM and APRI of the two groups were not statistically significant. After treatment, the liver fibrosis 4 items, liver function and cholestasis-related markers, LSM, and APRI were all significantly decreased in both groups, and the indexes in the Li-Dan-He-Ji group were significantly lower than those in the conventional treatment group [HA (ng/L): 165.81±21.57 vs. 203.87±25.88, PC III (μg/L): 69.86±9.32 vs. 81.82±7.39, IV-C (μg/L): 204.14±38.97 vs. 239.08±24.93, LN (μg/L): 162.40±17.39 vs. 190.86±15.97, TBil (μmol/L): 37.58±27.63 vs. 53.06±45.09, DBil (μmol/L): 20.55±19.34 vs. 30.08±27.39, ALP (U/L): 436.50±217.58 vs. 469.60±291.69, γ-GGT (U/L): 66.78±35.84 vs. 87.00±32.82, ALT (U/L): 64.75±50.53 vs. 75.20±50.19, AST (U/L): 77.25±54.23 vs. 96.80±59.77, TBA (μmol/L): 74.35±44.44 vs. 85.45±39.50, LSM (kPa): 5.24±0.39 vs. 7.53±3.16, APRI: 0.52±0.39 vs. 0.98±0.29, all P < 0.05]. After treatment, MDA in the two groups were significantly lower than those before treatment, and SOD and GSH were significantly higher than those before treatment. The level of SOD in the Li-Dan-He-Ji group was significantly higher than that in the conventional treatment group (kU/L: 64.56±6.69 vs. 51.58±5.98, P < 0.05). In addition, the liver retraction time (day: 20.13±10.97 vs. 24.33±13.46) and spleen retraction time (day: 25.93±13.01 vs. 29.14±14.52) in the Li-Dan-He-Ji group were significantly shorter than those in the conventional treatment group (both P < 0.05). CONCLUSIONS The use of Li-Dan-He-Ji in the treatment of cholestatic hepatic fibrosis can effectively improve the indicators of cholestasis, hepatic fibrosis, oxidative stress and clinical symptoms in children.
Child ; Humans ; Prospective Studies ; Cholestasis/pathology* ; Liver ; Liver Cirrhosis/drug therapy* ; Bilirubin/pharmacology* ; Oxidative Stress ; Aspartate Aminotransferases/metabolism* ; Superoxide Dismutase/metabolism*

Objective: To investigate the clinical value of serum glypican-3 (GPC3) detection in predicting recurrence of primary hepatocellular carcinoma (HCC). Methods: Through univariate and multivariate logistic regression analysis, the patients pathologically diagnosed with HCC in our hospital from March 2019 to January 2021 were enrolled as the experimental group (n=113), and patients with follow-up time longer than 6 months were included in the prognosis group(n=64). At the same time,20 healthy individuals and 20 individuals with benign liver disease from the physical examination center were enrolled by simple random sampling as control group (n=40). The serum GPC3 and alpha-fetoprotein (AFP) levels were respectively detected by ELISA and chemiluminescence. Then, the study explored the influential factors of the recurrence in HCC patients and constructed the HCC-GPC3 recurrence predicting model by logistic regression. Results: In the research, the sensitivity of GPC3 for the diagnosis of HCC was 61.95% (70/113) and AFP was 52.21% (59/113), meanwhile, the specificity of GPC3 could reach 87.50% (35/40) and AFP was 90.00% (36/40),respectively; The serum GPC3 levels of HCC patients with progressive stage, tumor size≥3 cm, vascular cancer thrombosis and portal venous thromboembolism were significantly higher than that of HCC patients with early stage, tumor size<3 cm, vascular cancer thrombosis and portal venous thromboembolism (Z=2.677, 2.848, 2.995, 2.252, P<0.05), independent of different ages, presence or absence of ascites, peritoneal metastasis, cirrhosis, intrahepatic metastasis (Z=-1.535, 1.011, 0.963, 0.394, 1.510, P>0.05), respectively. Univariate analysis showed that there were no statistically significant differences between the recurrence group and the non-recurrence group in terms of different age, tumor size, presence or absence of vascular cancer thrombosis, ascites, peritoneal metastasis, cirrhosis and AFP levels (χ²=2.012, 0.119, 2.363, 1.041, 0.318, 0.360, Z=0.748, P>0.05); The ratio of those with the progressive stage, portal venous thromboembolism and intrahepatic metastasis and GPC3 levels were all higher in the recurrence group than in the non-recurrence group (χ²=4.338, 11.90, 4.338, Z=2.805, P<0.05).Including the above risk factors in the logistic regression model, the logistic regression analysis showed that the stage, the presence of portal venous thromboembolism,intrahepatic metastasis and GPC3 levels were correlated with the prognosis recurrence of HCC patients (Wald χ² =4.421, 5.681, 4.995, 4.319, P<0.05), and the HCC-GPC3 recurrence model was obtained as: OcScore=-2.858+1.563×[stage]+1.664×[intrahepatic metastasis]+2.942×[ portal venous thromboembolism]+0.776×[GPC3]. According to the receiver operating characteristic curve(ROC), the area under the curve(AUC)of the HCC-GPC3 prognostic model was 0.862, which was better than that of GPC3 alone (AUC=0.704). The cut-off value of model SCORE was 0.699 (the cut-off value of GPC3 was 0.257 mg/L), furthermore, the total sensitivity and specificity of model were 83.3% and 82.4%, which were better than those of GPC3(60.0% and 79.4%).Kaplan-Meier showed that the median PFS was significantly shorter in HCC patients with high GPC3 levels (≥0.257 mg/L) and high values of the model SCORE (≥0.700) (χ²=12.73, 28.16, P<0.05). Conclusion: Besides diagnosing of HCC, GPC3 can may be an independent risk indicator for the recurrence of HCC and can more efficiently predicting the recurrence of HCC patients when combined with the stage, the presence or absence of intrahepatic metastasis and portal venous thromboembolism.
Humans ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/diagnosis* ; alpha-Fetoproteins/analysis* ; Biomarkers, Tumor ; Glypicans ; Ascites ; Venous Thromboembolism ; Peritoneal Neoplasms ; Liver Cirrhosis

Objective: To analyze the hepatic pathological characteristics and factors influencing an alanine transaminase value below twice the upper limit of normal in patients with chronic hepatitis B (CHB) and further explore the optimal ALT threshold strategy for initiating antiviral therapy. Methods: Clinical data of treatment-naïve CHB patients who underwent liver biopsies from January 2010 to December 2019 were retrospectively collected. Multiple regression models were used to explore the ALT levels and significant risk of hepatic histological changes (≥G2/S2). Receiver operating characteristic curve was used to evaluate the value of different models in diagnosing liver tissue inflammation≥G2 or fibrosis ≥ S2. Results: A total of 447 eligible CHB patients, with a median age of 38.0 years and 72.9% males, were included. During ALT normalization, there was significant liver inflammation (≥G2) and fibrosis (≥S2) in 66.9% and 53.0% of patients, respectively. With an ALT rise of 1-2×ULN, the proportions of liver inflammation≥G2 and fibrosis≥S2 were 81.2% and 60.0%, respectively. After adjusting for confounding factors, higher ALT levels (> 29 U/L) were found to be associated with significant liver inflammation (OR: 2.30, 95% CI: 1.11 ~ 4.77) and fibrosis (OR: 1.84, 95% CI: 1.10 ~ 3.09). After the measurement of glutamyltransferase-platelet ratio (GPR), the proportion of CHB patients with≥G2/S2 was significantly reduced under different treatment thresholds of ALT standards, and in particular, the erroneous evaluation of liver fibrosis≥S2 was significantly improved (33.5% to 57.5%). Conclusion: More than half of CHB patients have a normal ALT or one within 2 × ULN, regardless of whether or not there is apparent inflammation and fibrosis. GPR can significantly improve the precise assessment of different conditions of treatment thresholds for the ALT value in CHB patients.
Male ; Humans ; Adult ; Female ; Hepatitis B, Chronic/complications* ; Alanine Transaminase ; Retrospective Studies ; Liver/pathology* ; Liver Cirrhosis/complications* ; Inflammation/pathology* ; Hepatitis B e Antigens